Detoxification of herbicide/dioxin contaminated soil based on microbial diversity and their gene expression

2ND Meeting of The Agent Orange Working Group Detoxification of herbicide/dioxin contaminated soil based on microbial diversity and their gene expression Assc Prof Dr Dang Thi Cam Ha Institute of Biotechnology (IBT) Vietnam Academy of Science and Technology (VAST) Hanoi July 2, 2010 DANANG “HOT SPOT’ Composition of contaminants in former military soil • Herbicide: 2,4,5-T: 2,4-D/ 50 : 50 (20 100 – 000 000 µg/kg); • 2,3,7,8-TCDD: >99%, several thousands to hundred thousands pgTEQ/g ; • Other contaminants: TCP, DCP, PAHs etc.; • Heavy compounds of diesel oil Các nguồn vào/ra Đất “hộp đen” chất diệt cỏ khác v.v Sinh khốibiomass -Đa dạng VSV đất mức độ nào? - Microbial diversity? - Bằng cách đánh giá đa dạng VSV đất? Dection of this diversity by what kind of methods? Các thị sinh học bioindicators Vi sinh vật không nuôi cấy được-uncultural microbes Các vi sinh vật nuôi cấy –cultural microbes DNA/RNA Đa dạng VSV: đa dạng loài VSV khả trao đổi chất VSV Các nguồn 0,01-10% tổng số VSV -Total microbes Microbial diversity: species and their metobolisms Biodegradation pathways for detoxification of herbicide/dioxin contaminated sites • Oxidation • Declorination oxy hóa cắt vòng thơm Loại clo • Catalyzation (extracellular enzymes: Laccase + O2, MnP and LiP + H2O2) Xúc tác (enzyme ngoại bào: Laccase+O2, MnP LiP + H2O2) • Reductive declorination Loại khử clo không cần O Bioremediation Stimulation Kích thích sinh học Augmentation Tăng cường sinh học Phytoremediation Xử lý thực vật All resolutions based on microbial activities: oxydation, dehalogenation and catalysis reaction Tất giải pháp dựa hoạt động VSV tham gia oxy hóa cắt vòng, loại khử clo phản ứng xúc tác enzyme There are six biotechnologies which may be engineered for detoxification of soil contaminated with herbicides and dioxin and mixtures of other toxicants • Aerobic bioreactor followed by secure landfill for sensitive land uses requiring very low cleanup criteria; • Anaerobic bioreactor for chlorinated pesticides; • Anaerobic bioreactor followed by aerobic bioreactor for fast mineralization of certain contaminant mixtures (anaerobic/aerobic cycling); • Aerobic bioreactor, either biostimulated or bioaugmented to achieve cleanup levels rapidly; • Phytoremediation, either by passive land treatment or monitored natural attenuation; • Combination of all technologies, depending on site characterization and the contaminated properties In Vietnam: Active landfill for huge complex contaminated site was developed Microbial diversity of contaminated site and biotreatments (Culturable and unculturable) Herbicide/dioxin, DDT,HCH,TNT degrading microbes 45 bacterial strains • 12 Actinomycete strains • 20 Filamentous fungal strains • 20 Anaerobic communities • What have been researching and developing in IBTVAST concerning to the use of bioremediation for detoxification of herbicide/dioxin and other POP contaminated soil and sediment? POP (2,3,7,8-TCDD is included) degrading microbes and their degradation capability analyzed by HPLC, GC/MS, DR-Calux and other methods; Microbial community structure of contaminated soil and sediment (Lakes) and their change during bioremediation treatments of Danang and Bien Hoa former military bases; Characterization of bacteria, Actinomyces, filamentous fungi and some their functional genes (in a international GenBank database with author names: Identification of , oxidoreductase, peroxidase enzymes from different POP degrading microbes, consortium and whole community Detection of genus Dehalococcoides in different original HDIX sediments and almost biotreatments at the field trail at Danang and Bien Hoa sites These anaerobic microbes have been known with the name “dechlorinating workers” Summary IBT-VAST's activity • At laboratory scale: 32- 71% removal after a month • Field trial without supplied oxygen (0.5 - 1.5 m3): 51, 7% of initial concentration 268 ppb and 90 % (with lower TEQ) after two year treatment • Active landfill (10 and 100 m3): 70 % after two years • Anaerobic and aerobic bioreactors individually and together: 44,1% after only several months Vietnam- US cooperation in Danang Application of bioremediation at pilot scale (2m3) in Da Nang (6month evaluation) by VAST and EPA Vietnam Dang Thi Cam Ha Nguyen Ba Huu Nguyen Nguyen Quang Nguyen Quang Huy Dam Thuy Hang Nguyen Ngoc Anh Phung Khac Huy Chu USA Harry L Allen Vance Fong Acknowledgment – cảm ơn • • • • • • • • Ford foundation- Quỹ Ford Office 33- Văn phòng 33 US EPA US Embassy – Sứ quán Hoa Kỳ Mr Andrew Herrup Nguyen Thanh Tu VAST–Viện Khoa học & Công nghệ Việt Nam IBT- Viện Công nghệ sinh học Difficulty in evaluation of PCDDs/PCDFs concentration or toxicity What is a reason? • Dioxin distributed in the soil matrix is hetegenous Probably, “Black small pieces” came from heavy fraction of DO (resin and asphentence) contain more dioxin How to solve? Khắc phục • Use GC/MS ( scan method ) look for chemical composition change or biodegradation products • Analyze 3-5 samples for each detected soil at one time • Use small net of bolt for separation into parts and analyze parallel • Chemically analysis will be carried out only each months • Use the same soil for dioxin analysis in several laboratories Da Nang Pilot Study VAST/EPA Joint Bioremediation Study Bio -Technologies Tested: • Anaerobic Treatment Cells • Unamended “Anaerobic” Reference Cell • Aerobic (Oxidative) Land Treatment Units (LTU) • Unamended Open Reference Cell (Watered to prevent dehydration, but aerobic and subject to Natural Attenuation) System was managed by VAST personnel, who deserve gratitude for their hard work, diligence, and care Thanks to the VN Ministry of Defense for providing field support Product amendment - IBT - Vietnam Chemical amendment - EPA - US Chemical, physical and biological parameters for evaluation of herbicide/dioxin degradation • Total toxicity and congener toxicity (GC/MS, DR- Calux) • Concentration of 2,4,5-T, 2,4-D • Chemical composition change during bioremediation (GC/MS -Scan) • Moisture, pH • Microbial enumeration during treatment (cultivation methods in specific media + soil extract) • Evaluation of microbial community structure during treatment (PCR-DGGE by using 16S rRNA, 18S rRNA coding genes ) • Detection of some functional genes such as catechol 2,3 dioxygenase gene –C230, rdh gene v.v • Laccase producing microbes, • Dominated dioxin degraders Overall Summary of Pilot Study Results Degradation of TCDD Showing Confidence Intervals Conclusions and Recommendations • This study builds on other work conducted in Vietnam on bioremediation of TCDD and Agent Orange in the laboratory and in the field.* *A rich body of knowledge exists on bioremediation, including published and unpublished research results, which may be accessed by contacting VAST directly • The bioremediation pilot study has demonstrated that treatment by bioremediation is a practical alternative for reducing the concentration of TCDD in Da Nang soil to meet Vietnam’s cleanup goal of 1,000 parts per trillion (ppt.) It is estimated that the starting concentration of well-mixed soil and sediment in Da Nang would be approximately 9,900 ppt • Biodegradation was observed in both aerobic and anaerobic treatment tests Individual treatment recipes and treatment unit management schemes yielded different rates • The biodegradation rate may be linear, and if so, the amendment recipes demonstrated in this study are capable of reducing TCDD at an average rate of at least 100 parts per trillion (ppt.) per day This means a stockpile of excavated soil with average TCDD concentration of 10,000 ppt could be detoxified in a matter of months to achieve 1,000 ppt Anaerobic bioremediation would take about twice as long Conclusions and Recommendations • Aerobic biodegradation rate might be first-order, with a fixed percent per day being degraded In this case, total remediation time would not exceed years • The pilot study yielded important information relating to the engineering design factors, including rate, extent and growth conditions, which may be used for scaling up to full scale bioremediation • Conditions suitable for biodegradation as presented in this study may be established readily in an active landfill, which may be designed as an aerobic, anaerobic, or combination system • This study focused on measuring the reduction in dioxin concentration using chemical analyses in accordance with standard USEPA analytical methods Other analytical methods support the chemical results • Monitoring microbial diversity during the course of treatment is effective for corroborating chemical results Changes in the microbial community structure serve as indicators of change in growth conditions within the treatment unit, which could in turn facilitate dioxin biodegradation Conclusions and Recommendations • Bioaugmentation with small amounts of treated soil or contaminated sediment may be effective However, if suitable growth conditions are provided, the indigenous microbes in the mixed soil and sediment at Da Nang appear capable of degrading TCDD without bioaugmentation • Bioremediation is recognized as a “Green Technology” or “white Biotechnology” which has a very low energy requirement and produces few emissions Bioremediation is a permanent solution which produces a soil which can be returned to beneficial use • Knowledge gained from this project by both Vietnamese and US scientists will allow for design of customized recipes suitable for addressing dioxin and other persistent organic pollution problems throughout Vietnam and elsewhere • Data quality was enhanced by splitting samples among several laboratories * The variability in the data was independent of the laboratory *Contracted laboratories included SGS Systems in the US, a German dioxin laboratory, an MOD dioxin laboratory, and DR-CALUX Xin chân thành cảm ơn